The long-term objective of my research centers on elucidation of the mechanisms whereby extra-cellular signals are sensed by the cell cycle machinery and regulate cell cycle progression in normal versus tumor cells. This information will provide the necessary framework to elucidate how growth regulatory pathways are subverted in neoplasia. Our current studies focus on how growth-signaling pathways regulate the mitogenically responsive D-type cyclins and more specifically, how these pathways regulate accumulation of an active, nuclear cyclin D1-dependent kinase in normal versus cancerous cells. The noted overexpression of cyclin D1 in multiple human cancers highlights the importance of elucidating the mechanisms that regulate cyclin D1 activity. While cyclin D1 overexpression is a consequence of gene amplification and chromosome translocation in a subset of cancers, decreased cyclin D1 protein degradation, which depends on its phosphorylation on Thr286, is thought to be the key factor in a majority of cancers. Our progress in the characterization of this mechanism of cyclin D1 overexpression in cancer has been hindered by lack of information regarding the nature of the E3 ubiquitin ligase that directs cyclin D1 proteolysis. We have recently identified the SCF(Fbx4-(B crystallin) that controls cyclin D1 ubiquitination and degradation. This discovery has provided essential information and tools to assess the mechanisms that contribute to regulation of cyclin D1 accumulation during normal tissue development and the potential deregulation of cyclin D1 during malignant transformation. The identification of this E3 ligase as well as our recent preliminary studies lead to the overarching hypothesis that the SCF(Fbx4 -(B crystallin) ligase, via coordinated recognition of phospho- cyclin D1 by Fbx4 and (B crystallin, plays a critical role in the maintenance of cell growth and tissue maintenance. Experiments proposed in this grant will determine the contribution of Fbx4 to tissue homeostasis in vivo (Aim 1). We will utilize fbx4-/- cells to conclusively define the role of Fbx4 in the regulation of cyclin D1 and cell proliferation (Aim 2). The identification of residues in Fbx4 necessary for substrate recognition will be identified through characterization of mutant Fbx4 alleles identified in melanoma. PUBLIC HEALTH RELEVANCE: Overexpression of cyclin D1 in human cancer occurs frequently as a consequence of mutations in the machinery that destroys the cyclin D1 protein. In order to develop effective therapies that counter these events, it is necessary to identify proteins that direct cyclin D1 destruction and determine their role in the development of neoplasia. We have identified a critical component of the machinery, Fbx4, which directs destruction of the cyclin D1 protein, and the experiments described in this proposal will evaluate the biochemical and biological properties of Fbx4 with respect to human cancer.